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Isolation of cellular prion protein PrPc from brain homogenate and cerebrospinal fluid by immunoaffinity chromatography and structural analysis by 2-D map, immunoblot and by MS
Authors: Bílková Zuzana | Castagna Annalisa | Zanusso Gianluigi | Farinazzo Alessia | Monaco Salvatore | Viovy Jean-Louis | Righetti Pierre Giorgio
Year: 2003
Type of publication: ostatní - přednáška nebo poster
Name of source: Clinical Chemistry and Laboratory Medicine Special Supplement
Publisher name: Walter de Gruyter GmbH & Co. KG
Place: Berlin
Page from-to:
Titles:
Language Name Abstract Keywords
cze Izolace celulárního prionového proteinu z mozkového homogenátu a mozkomíšního moku pomocí imunoafinitní chromatografie a strukturní analýzy pomocí 2D elektroforézy, imunoblotu a MS imunosorbent, PrPc, magnetický reaktor
eng Isolation of cellular prion protein PrPc from brain homogenate and cerebrospinal fluid by immunoaffinity chromatography and structural analysis by 2-D map, immunoblot and by MS Introduction: the cellular prion protein (PrPc) is a glycoprotein abundant in neurons. PrPc represents the substrate for the generation of a conformational pathogenic isoform in prion diseases. The published detailed 2-D map of human PrPc both in brain and CSF demonstrated the unusual micro heterogeneity of this glycoprotein. To investigate more thoroughly this diversity it is necessary to obtain enough material to analyse all modified forms by MS. Aim was to prepare effective and gentle system for isolation, purification and concentration of prion protein in one step. Monoclonal antibodies are increasingly used to separate, isolate and purify complementary antigens. Immunoaffinity chromatography adds the selectivity and specificity of immunological reactions to the separation process. The goal was the construction of the immunosorbents to immobilise the antibody to the solid-phase support with magnetic core without adversely affecting the antibody function to capture. The magnetic core of the particles enables highly efficient and gentle separation during isolation and purification of PrP. Immobilisation via magnetic beads is particularly suitable for on-line immobilisation in a magnetically active microfluidic device (m-CHIP) directly coupled to MS. Results: 5B2 monoclonal mouse IgG 1 directed to a N-terminal epitope (residues 23-40) and 9H7 monoclonal mouse IgG 1 directed to a C-terminal epitope (residues 147-165) and polyclonal R029 rabbit antiserum were used. We aim in particular at high steric accessibility of the binding sites of antibodies, high operational and storage stability of binding activity, low non-specific sorption of protein to minimalise the contamination of the isolated antigen, and high recovery of antigen from immunosorbent. We prepared immobilised cellular prion protein for controlling possible interaction with other proteins in brain homogenate. The quality of the isolated protein and his heterogeneity were confirmed by SDS-PAGE, by immuno Immunosorbent,cellular prion protein PrPc, magnetic reaktor

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